Enantiomerically pure compounds (EPCs), especially amino and hydroxy acids as well as alcohols, amines, and lactones are increasingly useful in the pharmaceutical, food, and crop protection industries as building blocks for novel compounds not accessible through fermentation [1–4] as well as for asymmetric synthesis templates.[5–6] One very advantageous route to a wide variety of EPCs is the use of dehydrogenases, to afford either reduction of keto compounds or oxidation of alcohol or amine groups. The repertoire of dehydrogenases useful for synthesis of EPCs encompasses alcohol dehydrogenases (ADHs) [7], D- and L-lactate dehydrogenases (LDHs) [8], D- or L-hydroxyisocaproate dehydrogenases (D- or L-HicDHs) [9,10], or amino acid dehydrogenases such as leucine dehydrogenase (LeuDH) [10], phenylalanine dehydrogenase (PheDH) [11–13] or glutamate dehydrogenase (GluDH).[14] Monooxygenases have been used to synthesize, regio- and enantioselectively, lactones from cyclic ketones useful in the flavor and fragrance industries.[15]
Dehydrogenases and monooxygenases need nicotinamide-based cofactors, such as NAD+ and NADP+ or their reduced equivalents, NADH and NADPH, to function. Economic use of dehydrogenases and cofactor necessitates cofactor regeneration.[16] Cofactor costs, for example, $90 per gram for NAD+ have to be considered and having cofactors regenerated [17] would cut costs by the turnover number for such cofactors, between 100 and up to 600,000 [18].
Cofactor regeneration with alcohol dehydrogenases can be performed by using the same enzyme for in-situ substrate conversion and cofactor regeneration, usually employing isopropanol as co-substrate, as demonstrated with (S)-ADH from Thermoanaerobium brockii for both NADH and NADPH [19] and with (R)-ADH from L. brevis [20] for NADPH; this coupled-substrate approach, however, suffers from equilibrium limitations. The more common coupled-system approach, employing a separate second enzyme for regeneration, has been developed for reducing oxidized cofactors, NAD+ or NADP+, to NADH or NADPH. By far the most successful regeneration enzyme is formate dehydrogenase (FDH) for regeneration to either NADPH [24–25] or NADH, the latter even up to industrial scale [20–23]. Other options include the use of glucose 6-phosphate dehydrogenase [26] (to NADPH only) or of glucose dehydrogenase, GluDH [27–29]. For the opposite direction of regeneration, however, from NAD(P)H to oxidized cofactors NAD+ or NADP+, no universally accepted system exists.
There are some currently known NADH oxidases that are able to oxidize NADH to NAD+ with simultaneous reduction of O2 to either H2O2 or H2O [30–34]. Four-electron reduction to benign H2O is preferred over two-electron reduction to H2O2, which, even in small amounts, can deactivate either enzyme of the production-regeneration cycle. Addition of catalase as a possible remedy, to degrade the H2O2, increases complexity of the system to the point where three enzymes have to be coupled and adjusted as to their activity over time.
For reductive reactions with dehydrogenases or for monooxygenases, NAD(P)H has to be regenerated from NAD(P)+. For this problem, the system formate dehydrogenase (FDH)/formate is now used almost universally [35–37HCOOH+NAD+→NADH+H++CO2  (1)FDH functions as a universal regeneration enzyme in tandem with dehydrogenases catalyzing extremely enantioselective reduction reactions.[38–39]
For oxidative reactions requiring regeneration of NAD(P)+ from NAD(P)H, prior to the present invention, no universal cofactor regeneration system was known. Alcohol dehydrogenase (ADH) itself can be utilized to catalyze both the oxidative production reaction as well as the reductive regeneration reaction by adding isopropanol which is oxidized to acetone, but such a scheme tends to be equilibrium-limited and plagued by deactivation of ADH.[40] Both the ADH and the lactate dehydrogenase (LDH) systems [41] cannot take NADPH, in contrast to glutamate dehydrogenase (GluDH), which has been utilized to reduce α-ketoglutarate to L-glutamate.[42,43] NADH oxidases from thermophiles have been employed which regenerate NAD+ from NADH by reducing O2 to H2O2.[44]
What is needed are enzymes that regenerate NAD(P)H to oxidized cofactors NAD+ and NADP+ and synthesis methods that employ such enzymes alone or in coupled reactions. What is also needed are enzymes that perform the oxidation of NADH to NAD+ with the concomitant reduction of molecular oxygen to water as a solution to the cofactor regeneration problem from NADH to NAD+. Further, what is needed are methods for efficiently isolating the enzymes.